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Loss of p53 function by inactivating mutations results in abrogation of NO·-induced apoptosis in human lymphoblastoid cells. Here we report characterization of apoptotic signaling pathways activated by NO· in these cells by cDNA microarray expression and immunoblotting. A p53-mediated transcriptional response to NO· was observed in p53-wild-type TK6, but not in closely related p53-mutant WTK1, cells. Several previously characterized p53 target genes were up-regulated transcriptionally in TK6 cells, including phosphatase PPM1D (WIP1), oxidoreductase homolog PIG3, death receptor TNFRSF6 (Fas/CD95), and BH3-only proteins BBC3 (PUMA) and PMAIP1 (NOXA). NO· also modulated levels of several gene products in the mitochondria-dependent and death-receptor-mediated apoptotic pathways. Inhibitors of apoptosis proteins X-chromosome-linked inhibitor of apoptosis, cellular inhibitor of apoptosis protein-1, and survivin were significantly down-regulated in TK6 cells, but not in WTK1 cells. Smac release from mitochondria was induced in both cell types, but release of apoptosis-inducing factor and endonuclease G was detected only in TK6 cells. Fas/CD95 was increased, and levels of the antiapoptotic proteins Bcl-2 and Bcl-x/L were reduced in TK6 cells. Activation of procaspases 3, 8, 9, and 10, as well as Bid and poly(ADP-ribose) polymerase cleavage, were observed only in TK6 cells. NO· treatment did not alter levels of death receptors 4 and 5, Fas-associated death domain or proapoptotic Bax and Bak proteins in either cell line. Collectively, these data show that NO· exposure activated a complex network of responses leading to p53-dependent apoptosis via both mitochondrial and Fas receptor pathways, which were abrogated in the presence of mutant p53.

Supplementary Table 1 from Apoptotic Signaling Pathways Induced by Nitric Oxide in Human Lymphoblastoid Cells Expressing Wild-Type or Mutant p53

Supplementary Figure 1 from Apoptotic Signaling Pathways Induced by Nitric Oxide in Human Lymphoblastoid Cells Expressing Wild-Type or Mutant p53

Exposure to monocyclic aromatic alkylanilines (MAAs), namely 2,6-dimethylaniline (2,6-DMA), 3,5-dimethylaniline (3,5-DMA) and 3-ethylaniline (3-EA), was significantly and independently associated with bladder cancer incidence. 3,5-DMAP (3,5-dimethylaminophenol), a metabolite of 3,5-DMA, was shown to induce an imbalance in cytotoxicity cellular antioxidant/oxidant status, and DNA damage in mammalian cell lines. This study was designed to evaluate the protective effect of ascorbic acid (Asc) against the cytotoxicity, reactive oxygen species (ROS) production, genotoxicity and epigenetic changes induced by 3,5-DMAP in AA8 Chinese Hamster Ovary (CHO) cells. In different cellular fractions, 3,5-DMAP caused alterations in the enzyme activities orchestrating a cellular antioxidant balance, decreases in reduced glutathione levels and a cellular redox ratio as well as increases in lipid peroxidation and protein oxidation. We also suggest that the cellular stress caused by this particular alkylaniline leads to both genetic (Aprt mutagenesis) and epigenetic changes in histones 3 and 4 (H3 and H4). This may further cause molecular events triggering different pathological conditions and eventually cancer. In both cytoplasm and nucleus, Asc provided increases in 3,5-DMAP-reduced glutathione levels and cellular redox ratio and decreases in the lipid peroxidation and protein oxidation. Asc was also found to be protective against the genotoxic and epigenetic effects initiated by 3,5-DMAP. In addition, Asc supplied protection against the cell cycle (G1 phase) arrest induced by this particular alkylaniline metabolite.

Single-walled carbon nanotubes are particularly attractive for biomedical applications, because they exhibit a fluorescent signal in a spectral region where there is minimal interference from biological media. Although single-walled carbon nanotubes have been used as highly sensitive detectors for various compounds, their use as in vivo biomarkers requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we show that a polyethylene glycol ligated copolymer stabilizes near-infrared-fluorescent single-walled carbon nanotubes sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide concentration with a detection limit of 1 µM. The half-life for liver retention is 4 h, with sensors clearing the lungs within 2 h after injection, thus avoiding a dominant route of in vivo nanotoxicology. After localization within the liver, it is possible to follow the transient inflammation using nitric oxide as a marker and signalling molecule. To this end, we also report a spatial-spectral imaging algorithm to deconvolute fluorescence intensity and spatial information from measurements. Finally, we demonstrate that alginate-encapsulated single-walled carbon nanotubes can function as implantable inflammation sensors for nitric oxide detection, with no intrinsic immune reactivity or other adverse response for more than 400 days., Arnold and Mabel Beckman Foundation National Institute of Environmental Health Sciences: P30 ES002109 National Institutes of Health: ES007020 National Cancer Institute: P01 CA26731 2211, This work was supported by the National Institutes of Health (T32 Training Grant in Environmental Toxicology ES007020, to N.I.), the National Cancer Institute (grant P01 CA26731), the National Institute of Environmental Health Sciences (grant P30 ES002109), a Beckman Young Investigator Award and a National Science Foundation Presidential Early Career Award for Scientists and Engineers (to M.S.S.), the TUBITAK 2211 and 2214 Research fellowship programme (F.S. and S.S.) and the METU-DPT-OYP programme (F.S. and S.S). A Biomedical Innovation grant from Sanofi Aventis to M.S.S. is also acknowledged. The authors thank T. Grusecki for assistance with the deconvolution code, as well as R. Langer, D. Anderson and P. Dedon for helpful discussions.

Monocyclic aromatic amines are widespread environmental contaminants with multiple sources such as combustion products, pharmaceuticals, and pesticides. Their phenolic metabolites are converted intracellularly to electrophilic quinone imines upon autoxidation and can embed in the cellular matrix through a transimination reaction that leaves a redox-active residue as a substituent of lysine side-chain amino groups. To demonstrate the occurrence of this process within the cellular nucleus, Chinese hamster ovary AA8 cells were treated with the para-phenol of 3,5-dimethylamine, after which the histone proteins were isolated, derivatized, and subjected to tryptic digestion. The resulting peptides were analyzed by tandem mass spectrometry to determine which lysines were modified. Nine residues in histones H2A, H2B, and H4 were identified; these were located in histone tails, close to where DNA makes contact with the nuclear core particle, elsewhere on the protein surface, and deep within the core. Kinetics of disappearance of the modified lysines in cultured cells was determined using isotope-dilution mass spectrometry. AA8 cells were also transfected with the genetically encoded hydrogen peroxide biosensor HyPer in constructs that lead to expression of HyPer in different cellular compartments. Challenging the resulting cells with the dimethylaminophenol resulted in sustained fluorescence emission in each of the compartments, demonstrating ongoing production of H2O2. The kinetics of modified lysine loss determined by mass spectrometry was consistent with persistence of HyPer fluorescence emission. We conclude that the para-phenol of 3,5-dimethylamine can become stably integrated into the histone proteins, which are minimally repaired, if at all, and function as a persistent source of intracellular H2O2.

Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.

Dysregulated production of nitric oxide (NO•) and reactive oxygen species (ROS) by inflammatory cells in vivo may contribute to mutagenesis and carcinogenesis. Here, we compare cytotoxicity and mutagenicity induced by NO• and ROS in TK6 and AS52 cells, delivered by two methods: a well-characterized delivery system and a novel adaptation of a system for coculture. When exposed to preformed NO•, a cumulative dose of 620 μM min reduced the viability of TK6 cells at 24 h to 36% and increased mutation frequencies in the HPRT and TK1 genes to 7.7 × 10⁻⁶ (p0.05) and 24.8 × 10⁻⁶ (p0.01), 2.7- and 3.7-fold higher than background, respectively. In AS52 cells, cumulative doses of 1700 and 3700 μM min reduced viability to 49 and 22%, respectively, and increased the mutation frequency 10.2- and 14.6-fold higher than the argon control (132 × 10⁻⁶ and 190 × 10⁻⁶, respectively). These data show that TK6 cells were more sensitive than AS52 cells to killing by NO•. However, the two cell lines were very similar in relative susceptibility to mutagenesis; on the basis of fold increases in MF, average relative sensitivity values [(MF(exp)/MF(control))/cumulative NO• dose] were 5.16 × 10⁻³ and 4.97 × 10⁻³ μM⁻¹ min⁻¹ for TK6 cells and AS52 cells, respectively. When AS52 cells were exposed to reactive species generated by activated macrophages in the coculture system, cell killing was greatly reduced by the addition of NMA to the culture medium and was completely abrogated by combined additions of NMA and the superoxide scavenger Tiron, indicating the relative importance of NO• to loss of viability. Exposure in the coculture system for 48 h increased mutation frequency in the gpt gene by more than 9-fold, and NMA plus Tiron again completely prevented the response. Molecular analysis of gpt mutants induced by preformed NO• or by activated macrophages revealed that both doubled the frequency of gene inactivation (40% in induced vs 20% in spontaneous mutants). Sequencing showed that base-substitution mutations dominated the spectra, with transversions (30-40%) outnumbering transitions (10-20%). Virtually all mutations took place at guanine sites in the gene. G:C to T:A transversions accounted for about 30% of both spontaneous and induced mutations; G:C to A:T transitions amounted to 10-20% of mutants; insertions, small deletions, and multiple mutations were present at frequencies of 0-10%. Taken together, these results indicate that cell type and proximity to generator cells are critical determinants of cytotoxic and genotoxic responses induced by NO• and reactive species produced by activated macrophages.

The transcription factor NF-E2-related nuclear factor 2 (Nrf2) regulates expression of genes that protect cells from oxidative damage. Here, we characterized nitric oxide (•NO)-induced Nrf2–Kelch-like ECH-associated protein 1 (Keap1) signaling and its role in counteracting •NO-induced apoptosis of human colon cancer HCT116 cells. Nrf2 was localized in the cytoplasm in control cells; •NO triggered its rapid nuclear accumulation, transcriptional activation, and up-regulation of HO-1, NQO1, and GCL, but not GST A4 and P1 subunits. Nrf2 accumulation in the nucleus was also associated with enhanced transcription and posttranscriptional modifications. ( S )-nitrosation of Keap1 may contribute to nuclear accumulation of Nrf2 by facilitating its dissociation from Keap1, thus initiating •NO-mediated Nrf2–Keap1 signaling. •NO-mediated induction of ARE-dependent genes occurred well before apoptosis, as judged by caspase 3 activation. Collectively, these results show that the Nrf2–Keap1 signaling pathway mediates protective cellular responses to mitigate •NO-induced damage and may contribute to the relative resistance of HCT116 to •NO-induced cytotoxicity.

A major challenge in the synthesis of nanotube or nanowire sensors is to impart selective analyte binding through means other than covalent linkages, which compromise electronic and optical properties. We synthesized a 3,4-diaminophenyl-functionalized dextran (DAP-dex) wrapping for single-walled carbon nanotubes (SWNTs) that imparts rapid and selective fluorescence detection of nitric oxide (NO), a messenger for biological signalling. The near-infrared (nIR) fluorescence of SWNT(DAP-dex) is immediately and directly bleached by NO, but not by other reactive nitrogen and oxygen species. This bleaching is reversible and shown to be caused by electron transfer from the top of the valence band of the SWNT to the lowest unoccupied molecular orbital of NO. The resulting optical sensor is capable of real-time and spatially resolved detection of NO produced by stimulating NO synthase in macrophage cells. We also demonstrate the potential of the optical sensor for in vivo detection of NO in a mouse model.

We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10 9 bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d 9 ) dG-C8-ABP (MW = 443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H + ] +1 , at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444 → 328. The method was validated by analysis of DNA (100 μg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 μg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10 9 bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

Aromatic amines such as 2-naphthylamine and 4-aminobiphenyl are established human bladder carcinogens. Experimental evidence for carcinogenicity of monocylic aromatic amines is limited mostly to other organs, but a recent epidemiologic study of bladder cancer found that 2,6-dimethyl- (2,6-DMA), 3,5-dimethyl- (3,5-DMA), and 3-ethylaniline (3-EA) may play a significant role in the etiology of this disease in man. The present work was undertaken to test whether a genotoxic mechanism can account for the presumptive activity of 2,6-DMA, 3,5-DMA, and 3-EA by quantifying the binding of these compounds to DNA in vivo. Each of these three [(14)C]alkylanilines was administered at approximately 100 microg/kg to C57BL/6 mice, which were subsequently sacrificed 2, 4, 8, 16, and 24 h post-dosing. Bladder, colon, kidney, liver, lung, and pancreas were harvested from each animal, and DNA was isolated from each tissue. Adduct levels were determined by quantifying bound isotope using accelerator mass spectrometry. Adducts were detectable in the bladder and liver DNA samples from every animal at every time point at levels that ranged from 3 per 10(9) to 1.5 per 10(7) nucleotides. Adduct levels were highest in animals given 3,5-DMA and lowest in those given 3-EA. Levels in both bladder and liver declined by severalfold over the course of the experiment. Adducts were detected less frequently in the other four tissues. Taken together, the results strongly suggest that these three alkylanilines are metabolized in vivo to electrophilic intermediates that covalently bind to DNA and that adducts are formed in the DNA of bladder, which is a putative target organ for these alkylanilines.

Glutathione (GSH) plays an important role in cellular defense response in many in vitro and in vivo models. Here we investigated its role in NO -induced toxicity in cell culture and mouse models. Wild-type (TK6) and p53-null (NH32) human lymphoblastoid cells were treated with NO at a steady-state concentration of 0.6 μM, similar to the level estimated to occur in inflamed tissues. In both cell types, GSH was depleted by this exposure in a dose- and time-dependent manner. Contrary to expectations, prior depletion of GSH by treatment with l -buthionine- SR -sulfoximine did not potentiate NO -induced cell killing or DNA deamination in TK6 cells. In activated RAW264.7 murine macrophages producing NO , intracellular GSH content did not change, although γ-glutamate-cysteine ligase was upregulated. NO overproduction in RcsX lymphoma-bearing SJL mice resulted in significantly elevated GSH levels in various organs. Administration of the NO synthase inhibitor N -methylarginine abolished the increase in GSH in these animals. Collectively, these data indicate a multifaceted and complex involvement of GSH in responses of cells and tissues to toxic levels of NO . NO treatment effectively depleted GSH levels in human lymphoblastoid cells, but this alteration was not a critical initiating factor for NO -mediated toxicity. Murine macrophages maintained GSH homeostasis when exposed to endogenously produced NO . In RcsX lymphoma-bearing mice, upregulation of de novo synthesis of GSH appeared to be a response to the toxic effects of NO .

Antibodies are host proteins that comprise one of the principal effectors of the adaptive immune system. Their utility has been harnessed as they have been and continue to be used extensively as a diagnostic and research reagent. They are also becoming an important therapeutic tool in the clinician's armamentarium to treat disease. Antibodies are utilized for analysis, purification, and enrichment, and to mediate or modulate physiological responses. This overview of the structure and function of polyclonal and monoclonal antibodies describes features that distinguish one from the other. A limited review of their use as specific research, diagnostic, and therapeutic reagents and a list of printed and electronic resources that can be utilized to garner additional information on these topics are also included.

SIN-1 (3-morpholinosydnonimine), the active metabolite of the vasodilator drug molsidomine, decomposes spontaneously in solution. In the presence of oxygen, NO* and O(2)(*-) are released, generating peroxynitrite, a potent oxidizing agent, at a constant rate over a 2 h period. We utilized this system to investigate mechanisms of peroxynitrite-induced cytotoxicity, genotoxicity, apoptosis, and mitochondrial damage in two human lymphoblastoid cell lines carrying either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) genes. Treatment of TK6 cells with 5 mM SIN-1 for 1.5 h resulted in 28 +/- 6% survival 24 h later. Exposure in the presence of different radical scavengers significantly increased survival, as follows: cytochrome c, 96 +/- 3%; Tiron, 69 +/- 0%; SOD plus catalase, 83 +/- 5%; carboxy-PTIO, 87 +/- 3%; and uric acid, 87 +/- 2%. D-mannitol was ineffective in reducing lethality, as were SOD and catalase when added individually or in heat-inactivated form. Spontaneous as well as SIN-1-induced mutant fractions (MF) in both HPRT and TK genes were significantly higher in WTK-1 cells than in TK6 cells (p < 0.05-0.01). Exposure to 2.5 mM SIN-1 induced time-dependent apoptosis in TK6 cells, but not in WTK-1 cells. Mitochondrial membrane depolarization was also observed in both cell lines after SIN-1 treatment. Neutral comet assay demonstrated that SIN-1 treatment resulted in higher levels of DNA double-strand breaks in TK6 cells than in WTK-1 cells. Collectively, these data show that SIN-1 can be used as an effective peroxynitrite generator in cell culture experiments under these experimental conditions, in which it induced a greater apoptotic response but was less potent as a mutagen in TK6 cells compared with WTK-1 cells. Thus, p53 status was an important determinant of SIN-1 induced mutagenesis and apoptosis in these two human lymphoblastoid cell lines.

Most common alkylanilines in the environment are 2,6-dimethylaniline (2,6-DMA), 3,5-dimethylaniline (3,5-DMA), and 3-ethylaniline (3-EA). 3,5-Dimethylaminophenol (3,5-DMAP), a metabolite of 3,5-DMA, is of particular interest, as it is potentially genotoxic. Supplementation with organic or inorganic forms of selenium (Se) may reduce toxicity following exposure to a wide variety of environmental chemicals. This study was designed to evaluate the protective effects of sodium selenite (SS) and selenomethionine (SM) at varying time points of supplementation (24 h and 72 h) against the cytotoxicity, reactive oxygen species (ROS) production, and genotoxicity of 3,5-DMAP in CHO AS52 cells. 3,5-DMAP caused dose-dependent increase of cytotoxicity, ROS production and genotoxicity, and generated free radicals in the nuclei. Thioredoxin reductase (TrxR), catalase and glutathione reductase activities, and glutathione levels were significantly lower while lipid peroxidation and protein oxidation levels were higher after 3,5-DMAP treatment in both cytoplasm and the nucleus vs. control. After 24 h, both SS and SM provided protection in antioxidant/oxidant status of the 3,5-DMAP-treated cells; however other than supplying higher glutathione peroxidase and TrxR activities, 72 h supplementation did not provide advanced improvement. Selenocompounds may be beneficial against cytotoxic and genotoxic potential of 3,5-DMAP and might protect both nucleus and cytoplasm following exposure to alkylanilines.

Epidemiological studies have demonstrated extensive human exposure to the monocyclic aromatic amines, particularly to 3,5-dimethylaniline, and found an association between exposure to these compounds and risk for bladder cancer. Little is known about molecular mechanisms that might lead to the observed risk. We previously suggested that the hydroxylated 3,5-dimethylaniline metabolite, 3,5-dimethylaminophenol (3,5-DMAP), played a central role in effecting genetic change through the generation of reactive oxygen species (ROS) in a redox cycle with 3,5-dimethylquinoneimine. Experiments here characterize ROS generation by 3,5-DMAP exposure in nucleotide repair-proficient and -deficient Chinese hamster ovary cells as a function of time. Besides, various cellular responses discussed herein indicate that ROS production is the principal cause of cytotoxicity. Fluorescence microscopy of cells exposed to 3,5-DMAP confirmed that ROS production occurs in the nuclear compartment, as suggested by a previous study demonstrating covalent linkage between 3,5-DMAP and histones. 3,5-DMAP was also compared with 3,5-dimethylhydroquinone to determine whether substitution of one of the phenolic hydroxyl groups by an amino group had a significant effect on some of the investigated parameters. The comparatively much longer duration of observable ROS produced by 3,5-DMAP (7 vs. 1 day) provides further evidence that 3,5-DMAP becomes embedded in the cellular matrix in a form capable of continued redox cycling. 3,5-DMAP also induced dose-dependent increase of H2O2 and center dot OH, which were determined as the major free radicals contributing to the cytotoxicity and apoptosis mediated via caspase-3 activation. Overall, this study provides insight into the progression of alkylaniline-induced toxicity.

Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosome-dependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death.

One possible mechanism linking inflammation with cancer involves the generation of reactive oxygen, nitrogen, and halogen species by activated macrophages and neutrophils infiltrating sites of infection or tissue damage, with these chemical mediators causing damage that ultimately leads to cell death and mutation. To determine the most biologically deleterious chemistries of inflammation, we previously assessed products across the spectrum of DNA damage arising in inflamed tissues in the SJL mouse model nitric oxide overproduction ( Pang et al. ( 2007 ) Carcinogenesis 28 , 1807 - 1813 ). Among the anticipated DNA damage chemistries, we observed significant changes only in lipid peroxidation-derived etheno adducts. We have now developed an isotope-dilution, liquid chromatography-coupled, tandem quadrupole mass spectrometric method to quantify representative species across the spectrum of RNA damage products predicted to arise at sites of inflammation, including nucleobase deamination (xanthosine and inosine), oxidation (8-oxoguanosine), and alkylation (1,N(6)-ethenoadenosine). Application of the method to the liver, spleen, and kidney from the SJL mouse model revealed generally higher levels of oxidative background RNA damage than was observed in DNA in control mice. However, compared to control mice, RcsX treatment to induce nitric oxide overproduction resulted in significant increases only in inosine and only in the spleen. Further, the nitric oxide synthase inhibitor, N-methylarginine, did not significantly affect the levels of inosine in control and RcsX-treated mice. The differences between DNA and RNA damage in the same animal model of inflammation point to possible influences from DNA repair, RcsX-induced alterations in adenosine deaminase activity, and differential accessibility of DNA and RNA to reactive oxygen and nitrogen species as determinants of nucleic acid damage during inflammation.

The objective of this study was to compare monoclonal antibody production in hollow fiber bioreactor systems and murine ascites to determine the feasibility of the bioreactor system as a potential alternative to the use of mice. Three hybridoma cell lines were grown in each of three different hollow fiber bioreactor systems and in groups of 20 mice. Mice were primed with 0.5 ml pristane intraperitoneally 14 days prior to inoculation of 1X10(6) hybridoma cells. Each mouse was tapped a maximum of three times for collection of ascites. Ascites volumes and daily clinical observations were recorded. Bioreactors were harvested three times weekly for 65 day and were monitored by cell counts, cell viability and media glucose consumption. Time and materials logs were maintained. The total quantity of monoclonal antibody produced in 20 mice versus the mean production for the three different bioreactors in 65 days was as follows: cell line 2B11, 455 mg vs. 168 mg; cell line 3C9, 446 mg vs. 565 mg; and cell line RMK, 997 mg vs. 1023 mg. Mean monoclonal antibody concentration ranged from 4.07 to 8.37 mg/ml in murine ascites, and from 0.71 to 11.10 mg/ml in hollow fiber bioreactor system. Although time and material costs were generally greater for the bioreactors, these results suggest that hollow fiber bioreactor system merit further investigations as potentially viable in vitro alternatives to the use of mice for small scale (< 1 g) monoclonal antibody production.

Single-walled carbon nanotubes (SWNT) are particularly attractive for biomedical applications because they exhibit unique optical properties, fluorescing in the near infrared tissue transparency window, and allow for information transfer without the need for an internal power source. Although SWNT have been used as highly-sensitive detectors for various compounds, their use as in vivo biomarkers requires the simultaneous optimization of multiple parameters, including molecular recognition, biocompatibility, high fluorescence quantum efficiency and signal transduction. This work demonstrates two SWNT delivery mechanisms for in vivo detection of an inflammatory factor, nitric oxide (NO), that is associated with numerous diseases, including cancer, but is poorly understood due to its fast degradation rate and the lack of detection devices. First, a polyethylene glycol ligated copolymer is shown to stabilize SWNT sensors in physiological solutions, enabling an intravenous injection into mice. Second, a subcutaneous implant is investigated, with multiple SWNT concentrations, hydrogel shapes and sizes, and the gel’s physical properties examined to determine the preferred material for implantation. We found that both the injected and implanted SWNT are able to detect inflammation within an animal, thus providing the first demonstration of in vivo SWNT sensors as well as the first long term in vivo NO detection modality. This work demonstrates the ability of SWNT sensors to be utilized for in vivo data collection and possibly a wide range of biomedical applications.

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, the cellular HIF-1α level is regulated by hydroxylation by prolyl hydroxylases (PHDs), ubiquitylation, and proteasomal degradation. During hypoxia, degradation decreases, and its intracellular level is increased. Exogenously administered nitric oxide (NO)-donor drugs stabilize HIF-1α; thus, NO is suggested to mimic hypoxia. However, the role of low levels of endogenously produced NO generated during hypoxia in HIF-1α stabilization has not been defined. Here, we demonstrate that NO and reactive oxygen species (ROS) produced endogenously by human colon carcinoma HCT116 cells are responsible for HIF-1α accumulation in hypoxia. The antioxidant N-acetyl-L-cysteine (NAC) and NO synthase inhibitor N(G)-monomethyl L-arginine (L-NMMA) effectively reduced HIF-1α stabilization and decreased HIF-1α hydroxylation. These effects suggested that endogenous NO and ROS impaired PHD activity, which was confirmed by reversal of L-NMMA- and NAC-mediated effects in the presence of dimethyloxaloylglycine, a PHD inhibitor. Thiol reduction with dithiothreitol decreased HIF-1α stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favored its accumulation. This suggested that ROS- and NO-mediated HIF-1α stabilization involved S-nitrosation, which was confirmed by demonstrating increased S-nitrosation of PHD2 during hypoxia. Our results support a regulatory mechanism of HIF-1α during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity, probably by its S-nitrosation.

Several alkylanilines with structures more complex than toluidines have been associated epidemiologically with human cancer. Their mechanism of action remains largely undetermined, and there is no reported evidence that it replicates that of multicyclic aromatic amines even though the principal metabolic pathways of P450-mediated hydroxylation and phase II conjugation are very similar. As a means to elucidate their mechanisms of action, lethality and mutagenicity in the adenine phosphoribosyltransferase (aprt (+/-)) gene induced in several Chinese hamster ovary cell types by 2,6- and 3,5-dimethylaniline (2,6-DMA, 3,5-DMA) and their N- and ring-hydroxyl derivatives (N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP) were assessed. Dose-response relationships were determined in the parental AA8 cell line, its repair-deficient UV5 subclone and other repair-deficient 5P3NAT2 or -proficient 5P3NAT2R9 subclones engineered to express mouse cytochrome P4501A2 (CYP1A2) and human N-acetyltransferase (NAT2), and also in AS52 cells harboring the bacterial guanine-hypoxanthine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene of AS52 cells were characterized and found to be dominated by G:C to A:T and A:T to G:C transitions. Separately, treatment of AS52 cells with N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP, and 3,5-DMAP led to intracellular production of reactive oxygen species (ROS) for at least 24h after removal of the mutagens in every case. Using the comet assay, DNA strand breaks were observed in a dose-dependent manner in AS52 cells when treated with each of the four N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, and 3,5-DMAP derivatives. Comparative evaluation of the results indicates that the principal mechanism of mutagenic action is likely to be through redox cycling of intracellularly bound aminophenol/quinone imine structures to generate ROS rather than through formation of covalent DNA adducts.

Nitric oxide (NO) plays key roles in cell signaling and physiology, with diverse functions mediated by NO concentrations varying over three orders-of-magnitude. In spite of this critical concentration dependence, current approaches to NO delivery in vitro result in biologically irrelevant and poorly controlled levels, with hyperoxic conditions imposed by ambient air. To solve these problems, we developed a system for controlled delivery of NO and O[subscript 2] over large concentration ranges to mimic biological conditions. Here we describe the fabrication, operation and calibration of the delivery system. We then describe applications for delivery of NO and O[subscript 2] into cell culture media, with a comparison of experimental results and predictions from mass transfer models that predict the steady-state levels of various NO-derived reactive species. We also determined that components of culture media do not affect the steady-state levels of NO or O[subscript 2] in the device. This system provides critical control of NO delivery for in vitro models of NO biology and chemistry., National Cancer Institute (U.S.) (CA026731), National Cancer Institute (U.S.) (CA116318), National Institute of Environmental Health Sciences (ES002109)

Aflatoxin B (1) (AFB(1)) is a risk factor for hepatocellular carcinoma in humans. Infant, but not adult, mice are sensitive to AFB(1)-induced liver carcinogenesis; a single dose during the neonatal period leads to hepatocellular carcinoma in adulthood. Earlier work defined the mutational spectrum in the gpt gene of gpt delta B6C3F1 mice 3 weeks after exposure to aflatoxin. In the present study, we examined the gpt spectrum 10 weeks postdosing and expanded the study to examine, at 3 and 10 weeks, the spectrum at a second locus, the red/gam genes of the mouse λEG10 transgene. Whereas the gpt locus is typically used to define local base changes, the red/gam genes, via the Spi(-) assay, often are used to detect more global mutations such as large deletions and rearrangements. Three weeks after dosing with AFB(1), there was a 10-fold increase over the control in the Spi(-) mutant fraction (MF) in liver DNA; after 10 weeks, a further increase was observed. The MF in the gpt gene was also increased at 10 weeks compared with the MF at 3 weeks. No gender-specific differences were found in the Spi(-) or gpt MFs. Whereas Spi(-) mutations often signal large genetic changes, they did not in this specific case. The Spi(-) spectrum was dominated by GC to TA transversions, with one exceptionally strong hotspot at position 314. Using two genetic loci, the data show a strong preference for the induction of GC to TA mutations in mice, which is the dominant mutation seen in people exposed to aflatoxin.

Short Title: Aflatoxin adducts and mutation, Exposure to genotoxic chemicals at a young age increases cancer incidence later in life. Aflatoxin B[subscript 1] (AFB[subscript 1]) is a potent genotoxin that induces hepatocellular carcinoma (HCC) in many animal species and in humans. Whereas adult mice are insensitive to aflatoxin-induced carcinogenesis, mice treated with AFB[subscript 1] shortly after birth develop a high incidence of HCC in adulthood. Furthermore, the incidence of HCC in adult male mice treated as infants is much greater than in females, reasons for which are unclear. In this study, treatment with AFB[subscript 1] produced similar levels of DNA damage and mutations in the liver of newborn male and female gpt delta B6C3F1 mice. Twenty-four hours after dosing with AFB[subscript 1] (6 mg/kg), the highly mutagenic AFB1-FAPY adduct was present at twice the level of AFB[subscript 1]-N[superscript 7]-guanine in liver DNA of males and females. A multiple dose regimen (3 × 2 mg/kg), while delivering the same total dose, resulted in lower AFB1 adduct levels. Mutation frequencies in the gpt transgene in liver were increased by 20- to 30-fold. The most prominent mutations in AFB[subscript 1]-treated mice were G:C to T:A transversions and G:C to A:T transitions. At this 21-day time point, no significant differences were found in mutation frequency or types of mutations between males and females. These results show that infant male and female B6C3F1 mice experience similar amounts of DNA damage and mutation from AFB[subscript 1] that may initiate the neoplastic process. The gender difference in the subsequent development of HCC highlights the importance of elucidating additional factors that modulate HCC development., National Institutes of Health (U.S.) (R01 ES016313), National Institutes of Health (U.S.) (P30 ES002109), National Institutes of Health (U.S.) (P01 ES006052), National Institutes of Health (U.S.) (P30 ES003819)

Monoclonal antibodies and rabbit antisera were produced that recognized 4-aminobiphenyl, its major DNA adducts and other metabolites. The antigens used to raise these antibodies were synthesized by coupling the aromatic amine to protein through a diazotization reaction. The goal of this immunization strategy was to induce antibodies that also cross-reacted with most 4-aminobiphenyl-derived metabolites. A total of 20 mice and four rabbits were immunized and every animal produced a strong immune response for 4-aminobiphenyl and its derivatives. Two IgG1 monoclonal antibodies, 3D6 and 2E11, were isolated from two different mouse spleen cell fusions. One of the monoclonal antibodies, 3D6, had a high recognition for the three major 4-aminobiphenyl-DNA adducts: N-(deoxyguanosine-8-yl)-4-aminobiphenyl, N-(deoxyadenosin-8-yl)-4-aminobiphenyl and N-(deoxyguanosine-N2-yl)-4-aminobiphenyl, with affinity constants between 2 and 4 x 10(9) l/mol. In addition, one of the rabbit anti-sera had an affinity constant for the DNA adducts of 2.1 x 10(9) l/mole. Thus, the strategy to use a diazotization coupling reaction was successful at producing high-affinity aminobiphenyl-DNA adduct-specific antibodies. Preparative immunoaffinity resins were made for each monoclonal antibody. These resins quantitatively bound 500 ng each [3H]N-acetyl-aminobiphenyl, [3H]N-aminobiphenyl and [3H]N-(deoxyguanosine-8-yl)-4-aminobiphenyl. Preliminary experiments were performed to test the applicability of the preparative monoclonal antibody immunoaffinity column to isolate [3H]4-aminobiphenyl-derived metabolites in dosed rat and dog urine. About 70% of the radioactivity in rat or dog urine could be bound to the immunoaffinity columns. The combined immunoaffinity column/HPLC analysis of the dog urine led to the identification of a novel urinary metabolite, N-formyl-aminobiphenyl. HPLC analysis of a rat urine sample tentatively found 4-aminobiphenyl, N-acetyl-4-aminobiphenyl and N-formyl-4-aminobiphenyl by co-chromatography, and these compounds accounted for 20, 6.8 and 6.5% of the total radioactivity in the chromatogram respectively. Taken together, these data show that these 4-aminobiphenyl-specific monoclonal antibodies can be used in immunoaffinity columns to isolate metabolites and DNA adducts from biological samples.

Although many studies have focused on anticarcinogenic properties of capsaicin and resveratrol, molecular mechanisms by which they selectively induce apoptosis are incompletely characterized. We examined the role of nitric oxide (NO) and influence of p53 status during apoptosis induced by these agents in two isogenic HCT116 human colon carcinomas, wild-type p53 (p53-WT) and complete knockout of p53 (p53-null) cells. Capsaicin and resveratrol, alone or in combination, inhibited cell growth and promoted apoptosis by the elevation of NO; combined treatment in p53-WT cells was most effective. Increased NO production after treatment uniformly stimulated p53 and Bax expression through Mdm2 down-regulation in p53-WT cells, whereas all were unaffected in p53-null cells. Both cell types underwent a reduction in the levels of anti-apoptotic Bcl-2 protein, cytochrome c loss from mitochondria and activation of caspase 9 together with caspase 3, independently of p53 status. Concomitantly, we observed DR4, Fas(CD95) and caspase 8 activation, suggesting that these compounds activate both the mitochondrial and death receptor pathways working together to induce apoptosis. These findings provide insight into the mechanism of apoptotic action of capsaicin and resveratrol based on p53 status and indicate manipulation of NO may offer exciting opportunities to improve the effectiveness of colon cancer treatment.

Here we investigated the cytotoxicity of JS-K, a prodrug designed to release nitric oxide (NO•) following reaction with glutathione S-transferases, in multiple myeloma (MM). JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient-derived MM cells. JS-K induced apoptosis in MM cells, which was associated with PARP, caspase-8, and caspase-9 cleavage; increased Fas/CD95 expression; Mcl-1 cleavage; and Bcl-2 phosphorylation, as well as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) release. Moreover, JS-K overcame the survival advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells. Mechanistic studies revealed that JS-K–induced cytotoxicity was mediated via NO• in MM cells. Furthermore, JS-K induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by neutral comet assay, as well as H2AX, Chk2 and p53 phosphorylation. JS-K also activated c-Jun NH2-terminal kinase (JNK) in MM cells; conversely, inhibition of JNK markedly decreased JS-K–induced cytotoxicity. Importantly, bortezomib significantly enhanced JS-K–induced cytotoxicity. Finally, JS-K is well tolerated, inhibits tumor growth, and prolongs survival in a human MM xenograft mouse model. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.

Toxicity induced by nitric oxide (NO(*)) has been extensively investigated in many in vitro and in vivo experimental models. Recently, our laboratories found that both concentration and cumulative total dose are critical determinants of cell death caused by NO(*). Here, we report results of studies designed to define total dose thresholds and threshold effects for several NO(*)-induced toxicity and cellular responses and to determine impacts of p53 on them. We exposed human lymphoblastoid TK6 cells harboring wild-type p53 and isogenic p53-null NH32 cells to NO(*) delivered by a membrane delivery system. Cells were exposed at a steady state concentration of 0.6 microM for varying lengths of time to deliver increasing cumulative doses (expressed in units of microM min), and several end points of cytotoxicity and mutagenesis were quantified. Threshold doses for NO(*)-induced cytotoxicity were 150 microM min in TK6 cells and 300 microM min in NH32 cells, respectively. Threshold doses for NO(*)-induced apoptosis were identical to those for cytotoxicity, but mitochondrial depolarization thresholds were lower than those for cytotoxicity and apoptosis in both cell types. To gain insight into underlying mechanisms, cells of both types were exposed to sublethal (33% of cytotoxicity threshold), cytotoxicity threshold, or toxic (twice the cytotoxicity threshold) doses of NO(*). In TK6 cells (p53), the sublethal threshold dose induced DNA double-strand breaks, but nucleobase deamination products (xanthine, hypoxanthine, and uracil) in DNA were increased only modestly (50%) by toxic doses. Increased mutant fraction at the thymidine kinase gene (TK1) locus was observed only at the toxic dose of NO(*). Treatment of NH32 cells with NO(*) at the threshold or toxic dose elevated mutagenesis of the TK1 gene, but did not cause detectable levels of DNA double-strand breaks. At similar levels of cell viability, the frequency of DNA recombinational repair was higher in p53-null NH32 cells than in wild-type TK6 cells. NO(*) treatment induced p53-independent cell cycle arrest predominately at the S phase. Akt signaling pathway and antioxidant proteins were involved in the modulation of toxic responses of NO(*). These findings indicate that exposure to doses of NO(*) at or above the cytotoxicity threshold dose induces DNA double-strand breaks, mutagenesis, and protective cellular responses to NO(*) damage. Furthermore, recombinational repair of DNA may contribute to resistance to NO(*) toxicity and potentially increase the risk of mutagenesis. The p53 plays a central role in these responses in human lymphoblastoid cells.

Loss of p53 function by inactivating mutations results in abrogation of NO·-induced apoptosis in human lymphoblastoid cells. Here we report characterization of apoptotic signaling pathways activated by NO· in these cells by cDNA microarray expression and immunoblotting. A p53-mediated transcriptional response to NO· was observed in p53-wild-type TK6, but not in closely related p53-mutant WTK1, cells. Several previously characterized p53 target genes were up-regulated transcriptionally in TK6 cells, including phosphatase PPM1D (WIP1), oxidoreductase homolog PIG3, death receptor TNFRSF6 (Fas/CD95), and BH3-only proteins BBC3 (PUMA) and PMAIP1 (NOXA). NO· also modulated levels of several gene products in the mitochondria-dependent and death-receptor-mediated apoptotic pathways. Inhibitors of apoptosis proteins X-chromosome-linked inhibitor of apoptosis, cellular inhibitor of apoptosis protein-1, and survivin were significantly down-regulated in TK6 cells, but not in WTK1 cells. Smac release from mitochondria was induced in both cell types, but release of apoptosis-inducing factor and endonuclease G was detected only in TK6 cells. Fas/CD95 was increased, and levels of the antiapoptotic proteins Bcl-2 and Bcl-x/L were reduced in TK6 cells. Activation of procaspases 3, 8, 9, and 10, as well as Bid and poly(ADP-ribose) polymerase cleavage, were observed only in TK6 cells. NO· treatment did not alter levels of death receptors 4 and 5, Fas-associated death domain or proapoptotic Bax and Bak proteins in either cell line. Collectively, these data show that NO· exposure activated a complex network of responses leading to p53-dependent apoptosis via both mitochondrial and Fas receptor pathways, which were abrogated in the presence of mutant p53.

A novel delivery system was used to study NO-mediated cyto- and genotoxicity in two human lymphoblastoid cell lines, TK6 (wild-type p53) and NH32 (p53-null but isogenic to TK6). The delivery system, which supplied NO and O(2) continuously by diffusion through gas permeable tubing, was found to maintain the NO and O(2) concentrations at constant, predictable values. Cellular rates of NO and O(2) consumption and mass transfer coefficients for the two gases were measured in separate experiments and used to calculate the NO concentrations during exposure experiments. The TK6 and NH32 cells were each exposed to several steady state NO concentrations for varying lengths of time, so that the total dose (area under the concentration-time curve) covered a wide range. End point assays, including lethality, apoptosis, mitochondrial damage, and mutation rate in the thymidine kinase (TK1) gene locus, were performed at different posttreatment times. Control experiments using Ar instead of NO resulted in normal cell proliferation for all exposure times tested (up to 36 h). As compared to those controls, significant cell death, apoptosis, and mitochondrial membrane depolarization were observed in NO-treated TK6 cells, and the TK1 mutation rate was elevated. Of particular importance, toxic effects were observed only when the NO concentration and dose were greater than threshold values of approximately 0.5 micro M and approximately 150 micro M min, respectively. If neither or only one threshold was exceeded, the effects were insignificant; when both were exceeded, total cell survival and the number of nonapoptotic cells both decreased exponentially with increasing NO dose. In general, the NH32 cells were much more resistant to NO-induced damage and death than TK6 cells, demonstrating that p53 status is an important determinant of NO-induced cytotoxicity.

Hemochromatosis and Wilson disease (WD), characterized by the excess hepatic deposition of iron and copper, respectively, produce oxidative stress and increase the risk of liver cancer. Because the frequency of p53 mutated alleles in nontumorous human tissue may be a biomarker of oxyradical damage and identify individuals at increased cancer risk, we have determined the frequency of p53 mutated alleles in nontumorous liver tissue from WD and hemochromatosis patients. When compared with the liver samples from normal controls, higher frequencies of G:C to T:A transversions at codon 249 ( P < 0.001) and C:G to A:T transversions and C:G to T:A transitions at codon 250 ( P < 0.001 and P < 0.005) were found in liver tissue from WD cases, and a higher frequency of G:C to T:A transversions at codon 249 ( P < 0.05) also was found in liver tissue from hemochromatosis cases. Sixty percent of the WD and 28% of hemochromatosis cases also showed a higher expression of inducible nitric oxide synthase in the liver, which suggests nitric oxide as a source of increased oxidative stress. A high level of etheno-DNA adducts, formed from oxyradical-induced lipid peroxidation, in liver from WD and hemochromatosis patients has been reported previously. Therefore, we exposed a wild-type p53 TK-6 lymphoblastoid cell line to 4-hydroxynonenal, an unsaturated aldehyde involved in lipid peroxidation, and observed an increase in G to T transversions at p53 codon 249 (AGG to AGT). These results are consistent with the hypothesis that the generation of oxygen/nitrogen species and unsaturated aldehydes from iron and copper overload in hemochromatosis and WD causes mutations in the p53 tumor suppressor gene.

Melanoma patients experience inferior survival after bio-chemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS), and elevated levels of nitrotyrosine, a product derived from nitric oxide (NO). Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anti-cancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols decreased its cytotoxic potency. Activity of the pro-apoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/S-nitrisothiols, whereas lower activity resulted from cisplatin combined with stabilization of S-nitrisothiols. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2 (PHD2), the enzyme responsible for targeting the pro-survival transcription factor HIF-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy. Citation Format: Luiz C. Godoy, Chase C.T. Anderson, Rajdeep Chowdhury, Laura J. Trudel, Gerald N. Wogan. Melanoma fights cisplatin with NO: S-nitrosation as a mechanism supporting drug-resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 894. doi:10.1158/1538-7445.AM2013-894

Two analytical methods, one involving the combined use of reverse-phase HPLC and electrochemical detection (HPLC-EC) and one involving a mass spectrometric detection after gas chromatography separation (GC/MS), were developed for the detection of 8-oxoguanine in DNA. In order to obtain quantitative results, 2,6-diamino-8-oxopurine, whose chemical structure and electrochemical response are very similar to 8-oxoguanine, has been employed as an internal standard in the HPLC-EC assay. In the case of the GC/MS method, an isotopically stable (M + 4) 8-oxoguanine has been employed as an internal standard. Both methods are able to detect approximately 1 modification per 10(6) DNA bases. The background level of 8-oxoguanine in DNA as determined by GC/MS is approximately 50-fold higher than that determined by the HPLC-EC assay. The discrepancy between the two methods is due to an artifactual oxidation of guanine during the derivatization reaction as demonstrated by using pure guanine. The amount of 8-oxoguanine in guanine, determined by GC/MS, increases linearly with the time of derivatization, indicating that an oxidation occurs during the silylation reaction. Derivatization under nitrogen atmosphere reduces but does not suppress the artifactual oxidation. The amount of 8-oxoguanine in DNA, quantified by GC/MS, is comparable to that obtained by HPLC-EC when 8-oxoguanine is prepurified by HPLC or by immunoaffinity chromatography, prior to the silylation reaction. The artifactual formation of 8-oxoguanine during the derivatization reaction may explain, at least in part, why the values reported for 8-oxoguanine determination by GC/MS are generally about 1 order of magnitude higher than that determined by HPLC-EC. Prepurification of 8-oxoguanine from guanine is recommended in order to obtain reliable results by GC/MS which may be compared to HPLC-EC.

Exposure to nitric oxide (NO•) during inflammation has been implicated in the development of various forms of cancer, including melanoma. Clinical and epidemiologic studies indicate that expression of inducible nitric oxide synthase (NOS2), as well as NO• production by tumor cells, correlate with patient mortality. NO• favors resistance to drug-induced apoptosis in melanoma cells and may do so by caspases inactivation through S-nitrosation, which could represent a mechanism responsible for NO• inhibition of apoptosis. Here we further investigate how S-nitrosation impacts apoptotic and survival pathways in human melanoma. We demonstrate that A375, SK-Mel 100 and SK-Mel 28 cells constitutively express NOS2 and that an inhibitor of NO• synthesis decreases cell proliferation. Moreover, this same treatment, or the pre-incubation with the NO• scavenger carboxy-PTIO, leads to enhanced toxicity of cisplatin, suggesting that endogenous NO• regulates growth and enhances anti-apoptotic mechanisms in melanoma cells. This anti-apoptotic effect may be due to protein S-nitrosation promoted by endogenous NO•, because disruption of nitrosothiols by dithiothreitol promotes a remarkable increase in cisplatin-induced cell death in comparison with cells treated with cisplatin alone (80% vs. 20% cell death, respectively). On the other hand, when accumulation of cellular nitrosothiols is favored via inhibition of thioredoxin reductase by 1-chloro-2,4-dinitrobenzene, increased resistance to cisplatin is seen in A375 cells. Using the biotin switch technique (BST), a low, yet detectable level of endogenous protein S-nitrosation is seen in A375 cells. The properties conferred by exogenous NO• to melanoma cells were studied in a controlled-delivery system to simulate NO• concentrations known to occur in inflamed tissues. Using this system, the toxicity of NO• was shown to be lower in A375, SK-Mel 100 and SK-Mel 28 cells when compared to other non-melanoma cell lines. The possible protective effect of a chronic exposure to a non-toxic dose of exogenous NO• was investigated. A375 cells were treated with 0.64 µM NO• or argon (control) for 24 hours and then cisplatin was added to the system. Cell viability was assessed 24 hours later with trypan blue staining. While nearly 50% cell death was seen in argon+cisplatin-treated cells, no significant cell death occurred under NO•+cisplatin treatment, which points to an anti-apoptotic effect of low, sustained doses of NO• in this system. Finally, using the BST, the level of global protein S-nitrosation in the cells exposed to 0.64 µM NO• was shown to be considerably increased. Collectively, these results show that NO• favors survival and proliferation of melanoma cells in vitro, possibly by S-nitrosation of components of the signaling cascades. Identification of the proteins susceptible to this NO•-mediated modification will help to devise rational strategies for disease intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 204.

JS-K (O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) is a diazeniumdiolate class of prodrug which is designed to release nitric oxide (NO·) on reaction with glutathione S-transferases (GST). GST has been shown to be overexpressed in a broad spectrum of tumor cells. Therefore, JS-K can possibly turn GST overexpression to the tumor’s disadvantage by generating high intracellular concentrations of cytotoxic NO·. Multiple myeloma (MM) is currently an incurable hematological malignancy where new treatment options are urgently needed. In this study we investigated the cytotoxicity of JS-K in MM in vitro and in vivo. JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient MM cells (IC50: 0.3–2.5 mM). Importantly, no significant cytotoxic effects of JS-K at these doses were observed in normal peripheral blood mononuclear cells. JS-K treatment induced apoptosis in MM cells which was associated with PARP, caspase 8, and caspase 9 cleavage; increased cell surface expression of Fas/CD95; Mcl-1 cleavage; Bcl-2 phosphorylation; as well as mitochondrial cyt c, AIF, and EndoG release. Moreover, JS-K could overcome the survival and growth advantages conferred by exogenous IL-6 and IGF-1, or by adherence of MM cells to bone marrow stromal cells. Flow cytometry experiments revealed significant NO· generation in JS-K-treated MM cells. Since NO· is known to cause DNA double strand breaks (DSB), we hypothesized that JS-K induces DSB in MM cells and confirmed DSB formation by neutral comet assay. We further showed that JS-K also activated DNA damage response pathways as evidenced by H2AX, Chk2 and p53 phosphorylation. In addition, JNK was also activated by JS-K treatment in MM cells, and inhibition of JNK significantly decreased JS-K-induced cytotoxicity, suggesting that JS-K induced apoptosis is mediated via JNK signaling. Finally, JS-K was also significantly effective in inhibiting tumor growth and prolonging median survival (p < 0.01) in a human plasmacytoma xenograft mouse model. Analysis of tumors harvested from treated animals showed that JS-K induced apoptosis and decreased angiogenesis in vivo. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.

We describe, for the first time, the action of antibodies in anhydrous organic solvents. It has been demonstrated that the binding of a hapten, 4-aminobiphenyl, to the immobilized monoclonal antibody 2E11 is strong and specific not only in water but also in a variety of non-aqueous media. Further, the strength of interaction between antibody and hapten has been related to the hydrophobicity of the solvent: the more hydrophobic the solvent, the weaker the protein-ligand interaction.

A rapid and simple scheme has been developed for the isolation and purification of two of the major mutagenic heterocyclic amines formed in heated beef products by affinity chromatography using monoclonal antibodies which recognize 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Two cell lines producing IgG antibodies were established following fusion of Sp2 or P3x.63 myeloma cells with spleen cells of immunized BALB/cby mice. The antigen was bovine gamma globulin haptenized with 2-(3-carboxypropylthio)-3-methylimidazo-[4,5-f]quinoline. The antibodies were immobilized on CNBr-activated Sepharose 4B. IQ and MeIQx formed in heated beef products were partially purified by XAD-2 chromatography and then applied to the affinity columns. Purification by affinity chromatography was adequate for subsequent quantitative analysis by HPLC with UV detection. With this purification scheme as little as 1 g of beef extract or 15 g of fried beef could be assayed for IQ and MeIQx at the part per billion level. Both antibodies had similar affinity constants for IQ (9.3 X 10(6) and 6.7 X 10(6) M-1) and for MeIQx (7.1 X 10(5) and 2.7 X 10(5) M-1) and both were suitable for immunoaffinity purification of IQ from complex mixtures. MAb2 could be used as well to selectively remove MeIQx from meat products after partial purification by XAD-2. MAb1, despite having a 3-fold higher affinity than MAb2 for MeIQx, could not be used for affinity chromatography for this mutagen.

Monoclonal antibodies specific for aflatoxin B1, aflatoxin B2, aflatoxin M1, and the major aflatoxin-DNA adducts were obtained following fusion of mouse SP-2 myeloma cells with spleen cells of mice immunized with aflatoxin B1 covalently bound to bovine gamma globulin. The aflatoxin-modified protein used to immunize mice was produced chemically by activating aflatoxin B1 to a 2,3-epoxide derivative, which then covalently bound to the protein. One of the monoclonal antibodies isolated (2B11) was found to be a high-affinity IgM antibody with an affinity constant for aflatoxin B1, aflatoxin B2, and aflatoxin M1 of about 1 X 10(9) liters per mol. In a competitive radioimmunoassay using [3H]aflatoxin B1, 3 pmol (1 ng) of aflatoxin B1, aflatoxin B2, or aflatoxin M1 caused 50% inhibition with this antibody. The antibody also had significant cross-reactivity for the major aflatoxin-DNA adducts: 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 and 2,3-dihydro-2-(N5-formyl-2',5', 6'-triamino-4'oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1. The antibody was also covalently bound to Sepharose-4B and used in a column-based solid-phase immunosorbent assay system. Aflatoxins added in vitro to phosphate buffer, human urine, human serum, or human milk at levels expected to be obtained in human samples acquired from environmentally exposed individuals were quantitatively recovered by applying the mixture to this antibody affinity column purification system. Preliminary studies using urine samples from rats injected with radiolabeled aflatoxin B1 have also indicated that aflatoxin metabolites can be isolated by these methods. Furthermore, we have found that the monoclonal antibody affinity columns can be regenerated for multiple use. Therefore, the monoclonal antibodies and their application to affinity chromatography represents a useful and rapid technique to purify environmentally occurring levels of this carcinogen and some of its metabolites for quantitative measurements.